AIB1, a Steroid Receptor Coactivator Amplified in Breast and Ovarian Cancer

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An AIB1-specific primer, N8F1 (TCATCACTTCCGACAACAGAGG), was biotinylated and used to capture cDNA clones from a human lung cDNA library (Gibco, BRL) with the GENETRAPPER cDNA positive selection system (Gibco, BRL). The largest clone (5.8 kb), designated pCMVSPORT-B11, was selected for sequence analysis. To obtain full-length AIB1 sequence, we constructed a random-primed library from BT474 in bacteriophage λ-Zap (Stratagene) and hybridized it with a 372-base pair ³²P-labeled polymerase chain reaction (PCR) product amplified from a human spleen cDNA library by using primers designed from the 5′ sequence of pCMVSPORT-B11, PM-U2 (CCAGAAACGTCACTATCAAG), and Bll-llRA (TTACTGGAACCCCCATACC). Plasmid rescue of 19 positive clones yielded a clone, pBluescript-R22, that overlapped pCMVSPORT-B11 and contained the 5′ end of the coding region. To generate a full-length AIB1 clone, we subcloned the 4.85-kb Hind III–Xho I fragment of pCMVSPORT-B11 into the Hind III–Xho I sites of pBluescript-R22. We then subcloned the 4.84-kb Not I–Nhe I fragment of the full-length clone containing the entire coding region into the Not I–Xba I sites of the expression vector pcDNA3.1 (Invitrogen) and generated pcDNA3.1-AIB1.

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We obtained established breast cancer cell lines from the American Type Culture Collection (ATCC) (BT474, MCF-7, T-47D, MDA-MB-361, MDA-MB-468, BT-20, MDA-MB-436, and MDA-MB-453), the Arizona Cancer Center (UACC-812), and the National Cancer Institute (ZR75-1). We obtained ovarian cancer cell line BG-1 from Jeff Boyd, University of Pennsylvania. For Northern analysis, we obtained normal human mammary gland total RNA pooled from six individuals between 16 and 35 years old from Clontech. For FISH analysis, interphase nuclei were fixed in methanol and acetic acid (3:1) and dropped onto microscope slides.

Data not shown.

The BioPrime DNA labeling system (Gibco BRL) was used to label genomic clones containing either AIB1 (3) or the 20q13 amplicon (RMC20C001) (14) with Spectrum Orange deoxyuridine triphosphate (dUTP) (Vysis). We used 20q11 and 20p probes (University of California, Berkeley, Resource for Molecular Cytogenetics RMC20P037 and RMC20P039) as reference probes for two-color FISH analysis after labeling with biotin-16-dUTP (BMB) by nick-translation. FISH followed the method of Pinkel et al. (19) with minor modifications. Fluorescent images were captured with a Zeiss axiophot microscope equipped with a charge-coupled device camera and IP Lab Spectrum software (Signal Analytics). FISH analysis of uncultured breast cancer samples was performed as described (14) from either disaggregated nuclei or sections of ethanol-fixed tumors. Tumors were categorized into three groups according to AIB1 copy number: low (fewer than four copies per cell or no increased relative copy number), moderate (four to six copies per cell or 1.5- to 3-fold relative copy number), and high (more than six copies per cell or >3-fold relative copy number). Only high copy number increases were taken as evidence for gene amplification. In 105 unselected cases, 10 were scored high, 13 were moderate, and 82 were low. ER status was determined by immunohistochemistry for 83 of these specimens including 9 of the 10 cases with AIB1 amplification. Of these nine specimens, four were ER positive and five were ER negative.

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AIB1 mRNA in situ expression was determined by using three cDNA fragments (covering nucleotides 1733 to 2579, 3072 to 3580, and 3533 to 4120 of the AIB1 cDNA) labeled with deoxyadenosine [³³P]triphosphate by PCR. We hybridized ethanol-fixed sections from 75 primary breast tumors and six adjacent normal breast tissues as described (20). We knew the ER status for 66 of these samples. AIB1 was highly expressed in 24 of 44 (55%) of the ER-positive cases and in 8 of 22 (36%) of the ER-negative cases.

CV-1 cells (ATCC) were grown and maintained in phenol red–free Dulbecco's modified Eagle's medium supplemented with 10% charcoal-stripped fetal bovine serum. We plated cells into six-well culture dishes at 1.0 × 10⁵ cells per well and allowed them to grow overnight. Transfections were done with calcium phosphate coprecipitation (Clontech) according to the manufacturer's protocol. Transfected DNA included 5 ng of pRL-CMV internal control plasmid, 1.0 μg of ER reporter, 250 ng of pHEGO-hyg ER expression vector, the indicated amount of pcDNA3.1-AIB1, or an equivalent amount of pcDNA3.1 empty vector, and salmon sperm DNA to a total of 7 μg of DNA per dish. After transfection, we incubated cells in the absence or presence of 10 nM 17β-estradiol or 100 nM 4-OHT. Cell lysates were harvested and assayed 48 hours after transfection. We determined reporter activities with the dual-luciferase reporter assay system (Promega) and the results are expressed in relative luminescence units (RLU) (luciferase/Renilla luciferase). We obtained pRL-CMV and pGL3-promoter from Promega and pHEGO-hyg from ATCC. ER reporter pGL3.luc.3ERE, which contains three tandem copies of the ERE upstream from the simian virus 40 promoter driving the luciferase gene, was the kind gift of Fern Murdoch, Uniformed Services University of the Health Sciences.

We constructed GST fusion proteins by generating PCR fragments of AIB1 encoding amino acids 1 to 194 and amino acids 605 to 1294 inserted into pGEX 6P-2 (Pharmacia). GST pulldown analysis was done as described by Le Douarin (21) with the following modifications: 6.7 μg of ER (Panvera) was preincubated with or without 2 μM estradiol. Separately, we preincubated 10 μg of either GST, GST-AIB.N1 (containing amino acids 1 to 194), or GST-AIB.T1 (containing amino acids 605 to 1294) with preequilibrated glutathione-Sepharose (Pharmacia) and washed it with binding buffer. ER with or without estradiol mixture was incubated with GST fusion protein-glutathione-Sepharose mixture in binding buffer containing 0.1% bovine serum albumin for 1 hour at room temperature. Glutathione-Sepharose beads were washed five times in binding buffer, and bound proteins were eluted in SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and separated by SDS-PAGE. Separated proteins were transferred to nylon membranes, incubated sequentially with monoclonal antibody H-151 to ER (StressGen) and horseradish peroxidase containing goat anti-mouse immunoglobulin G Fc (Jackson Immunoresearch), and detected by chemiluminescence.

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Abbreviations for the amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

We obtained cDNA clones from Research Genetics [TIF2 (clone 132364, GenBank accession number ), SRC-1 (clone 418064, GenBank accession number )], ATCC (pHEGO-hyg, ATCC number 79995), or Clontech (β-actin). The AIB1 probe was a 2.2-kb Not I–Sac I fragment of pCMVSPORT-B11.

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The authors gratefully acknowledge the technical assistance of Nasser Parsa and Robin Veldman, Darryl Leja for preparation of the illustrations, and Roger Meisfeld for helpful discussions.